Abstract

Multipotential stromal cells/ mesenchymal stem cells (MSCs) can regenerate bone and adipose tissues, as these cells form osteoblasts and adipocytes. A major hurdle to using MSC however is cell-loss post-implantation. This cell death is partly due to inflammatory cytokines such as FasL generated in the implant site. In this study we found that FasL kills MSC by increasing reactive oxygen species in addition to activation of caspases. Thus we sought ways to protect MSC from FasL and other pro-death stimuli. Our group previously reported that soluble epidermal growth factor (sEGF) promotes MSC expansion but does not support survival from FasL. Tethering EGF onto a two-dimensional surface (tEGF) altered MSC responses, causing sustained cell-surface activation of EGFR, protecting from FasL. However, for tEGF to be useful in bone regeneration, it needs to allow for MSC differentiation into osteoblasts, while also protecting emerging osteoblasts. Our lab has also shown that the matrikine Tenascin-C binds EGFR with a low affinity but a high avidity, and we proposed that Tenascin-C should also lead to cell-surface signaling of EGFR, causing survival. We found that tEGF and Tenascin-C did not block induced differentiation of MSCs into osteoblasts, or adipocytes, a default MSC-differentiation pathway. tEGF protected differentiating osteoblasts from FasL mediated death. Differentiating adipocytes became resistant to FasL, with tEGF having no further survival effect. tEGF also protected MSC from combined insults of FasL, serum deprivation and hypoxia. Tenascin-C was found to protect MSC from FasL by activating sustained EGFR signaling from the cell surface. We also found that MSC exist in a state of pre-autophagy, with cells filled with early autophagosomes that are not degraded. These autophagosomes are lost as MSC differentiate into osteoblasts, suggesting that MSC exist in a state of preparedness, to form new protein during nutrient challenge. Our results suggest that MSCs and differentiating osteoblasts need protective signals to survive the inflammatory wound. tEGF or Tenascin-C can serve this function.